Packaging systems for human recombinant adenovirus to be used in gene therapy

ABSTRACT

Presented are ways to address the problem of replication-competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector are suited for large scale production of recombinant adenoviruses. A described system produces replication-defective adenovirus. The system includes a primary cell containing a nucleotide derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleotide is derived from an adenovirus, has at least one functional encapsidation signal and at least one functional Inverted Terminal Repeat, and lacks overlapping sequences with the nucleic acid of the cell. Otherwise, the overlapping sequences would enable homologous recombination leading to replication-competent adenovirus in the primary cell into which the isolated recombinant nucleotide is to be transferred.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of co-pending U.S. patentapplication Ser. No. 10/125,751, filed Apr. 18, 2002, U.S. Pat. No.______, which is a continuation of application Ser. No. 09/506,548,filed Feb. 16, 2000, now U.S. Pat. No. 6,602,706, issued Aug. 5, 2003,which is a division of Ser. No. 09/334,765, filed Jun. 16, 1999, nowU.S. Pat. No. 6,238,893, which is a continuation of Ser. No. 08/793,170,filed Mar. 25, 1997, now U.S. Pat. No. 5,994,128, issued Nov. 30, 1999,which is a national entry of PCT International Patent ApplicationPCT/NL96/00244, filed Jun. 14, 1996, which itself claims priority fromEuropean Patent Application EP 95201728.3, filed Jun. 26, 1995, andEuropean Patent Application EP 95201611.1, filed Jun. 15, 1995, thecontents of each of which are hereby incorporated herein by thisreference.

TECHNICAL FIELD

The invention relates generally to the field of biotechnology andrecombinant DNA technology, more in particular to the field of genetherapy. In particular, the invention relates to gene therapy usingmaterials derived from adenovirus, specifically human recombinantadenovirus. It especially relates to novel virus-derived vectors andnovel packaging cell lines for vectors based on adenoviruses.

BACKGROUND

Gene therapy is a recently developed concept for which a wide range ofapplications can be and have been envisioned. In gene therapy, amolecule carrying genetic information is introduced into some or allcells of a host, as a result of which the genetic information is addedto the host in a functional format.

The genetic information added may be a gene or a derivative of a gene,such as a cDNA, which encodes a protein. This is a functional format inthat the protein can be expressed by the machinery of the host cell.

The genetic information can also be a sequence of nucleotidescomplementary to a sequence of nucleotides (either DNA or RNA) presentin the host cell. This is a functional format in that the added DNA(nucleic acid) molecule or copies made thereof in situ are capable ofbase pairing with the complementary sequence present in the host cell.

Applications include the treatment of genetic disorders by supplementinga protein or other substance which, because of the genetic disorder, iseither absent or present in insufficient amounts in the host, thetreatment of tumors, and the treatment of other acquired diseases suchas (auto)immune diseases, infections, etc.

As may be inferred from the above, there are basically three differentapproaches in gene therapy: the first directed towards compensating fora deficiency in a (mammalian) host, the second directed towards theremoval or elimination of unwanted substances (organisms or cells) andthe third towards application of a recombinant vaccine (against tumorsor foreign micro-organisms).

For the purpose of gene therapy, adenoviruses carrying deletions havebeen proposed as suitable vehicles for genetic information. Adenovirusesare non-enveloped DNA viruses. Gene-transfer vectors derived fromadenoviruses (so-called “adenoviral vectors”) have a number of featuresthat make them particularly useful for gene transfer for such purposes.For example, the biology of the adenovirus is characterized in detail,the adenovirus is not associated with severe human pathology, theadenovirus is extremely efficient in introducing its DNA into the hostcell, the adenovirus can infect a wide variety of cells and has a broadhost-range, the adenovirus can be produced in large quantities withrelative ease, and the adenovirus can be rendered replication defectiveby deletions in the early-region 1 (“E1”) of the viral genome.

The adenovirus genome is a linear double-stranded DNA molecule ofapproximately 36,000 base pairs with the 55 kDa terminal proteincovalently bound to the 5′ terminus of each strand. The adenoviral(“Ad”) DNA contains identical Inverted Terminal Repeats (“ITR”) of about100 base pairs with the exact length depending on the serotype. Theviral origins of replication are located within the ITRs exactly at thegenome ends. DNA synthesis occurs in two stages. First, the replicationproceeds by strand displacement, generating a daughter duplex moleculeand a parental displaced strand. The displaced strand is single strandedand can form a so-called “panhandle” intermediate, which allowsreplication initiation and generation of a daughter duplex molecule.Alternatively, replication may proceed from both ends of the genomesimultaneously, obviating the requirement to form the panhandlestructure. The replication is summarized in FIG. 14 adapted from Lechnerand Kelly (1977).

During the productive infection cycle, the viral genes are expressed intwo phases: the early phase, which is the period up to viral DNAreplication, and the late phase, which coincides with the initiation ofviral DNA replication. During the early phase, only the early geneproducts, encoded by regions E1, E2, E3 and E4, are expressed, whichcarry out a number of functions that prepare the cell for synthesis ofviral structural proteins (Berk, 1986). During the late phase, the lateviral gene products are expressed in addition to the early geneproducts, and host cell DNA and protein synthesis are shut off.Consequently, the cell becomes dedicated to the production of viral DNAand of viral structural proteins (Tooze, 1981).

The E1 region of adenovirus is the first region of adenovirus expressedafter infection of the target cell. This region consists of twotranscriptional units, the E1A and E1B genes, which both are requiredfor oncogenic transformation of primary (embryonic) rodent cultures. Themain functions of the E1A gene products are 1) to induce quiescent cellsto enter the cell cycle and resume cellular DNA synthesis and 2) totranscriptionally activate the E1 B gene and the other early regions(E2, E3, E4). Transfection of primary cells with the E1A gene alone caninduce unlimited proliferation (immortalization) but does not result incomplete transformation. However, expression of E1A in most casesresults in induction of programmed cell death (apoptosis) and onlyoccasionally immortalization (Jochemsen et al., 1987). Co-expression ofthe E1B gene is required to prevent induction of apoptosis and forcomplete morphological transformation to occur. In established immortalcell lines, high level expression of E1A can cause completetransformation in the absence of E1B (Roberts et al., 1985).

The E1B-encoded proteins assist E1A in redirecting the cellularfunctions to allow viral replication. The E1B 55 kDa and E4 33 kDaproteins, which form a complex that is essentially localized in thenucleus, function in inhibiting the synthesis of host proteins and infacilitating the expression of viral genes. Their main influence is toestablish selective transport of viral mRNAs from the nucleus to thecytoplasm concomitantly with the onset of the late phase of infection.The E1B 21 kDa protein is important for correct temporal control of theproductive infection cycle, thereby preventing premature death of thehost cell before the virus life cycle has been completed. Mutant virusesincapable of expressing the E1B 21 kDa gene product exhibit a shortenedinfection cycle that is accompanied by excessive degradation of hostcell chromosomal DNA (deg-phenotype) and in an enhanced cytopathiceffect (cyt-phenotype) (Telling et al., 1994). The deg and cytphenotypes are suppressed when in addition the E1A gene is mutated,indicating that these phenotypes are a function of E1A (White et al.,1988). Furthermore, the E1B 21 kDa protein slows down the rate by whichE1A switches on the other viral genes. It is not yet known through whichmechanisms E1B 21 kDa quenches these E1A dependent functions.

Vectors derived from human adenoviruses, in which at least the E1 regionhas been deleted and replaced by a gene of interest, have been usedextensively for gene therapy experiments in the pre-clinical andclinical phase.

As stated before, all adenovirus vectors currently used in gene therapyare believed to have a deletion in the E1 region, where novel geneticinformation can be introduced. The E1 deletion renders the recombinantvirus replication defective (Stratford-Perricaudet and Perricaudet,1991). We have demonstrated that recombinant adenoviruses are able toefficiently transfer recombinant genes to the rat liver and airwayepithelium of rhesus monkeys (Bout et al., 1994b; Bout et al., 1994a).In addition, we (Vincent et al., 1996a; Vincent et al., 1996b) andothers (see, e.g., Haddada et al., 1993) have observed a very efficientin vivo adenovirus mediated gene transfer to a variety of tumor cells invitro and to solid tumors in animals models (lung tumors, glioma) andhuman xenografts in immunodeficient mice (lung) in vivo (reviewed byBlaese et al., 1995).

In contrast to (for instance) retroviruses, adenoviruses 1) do notintegrate into the host cell genome, 2) are able to infect non-dividingcells, and 3) are able to efficiently transfer recombinant genes in vivo(Brody and Crystal, 1994). Those features make adenoviruses attractivecandidates for in vivo gene transfer of, for instance, suicide orcytokine genes into tumor cells.

However, a problem associated with current recombinant adenovirustechnology is the possibility of unwanted generation ofreplication-competent adenovirus (“RCA”) during the production ofrecombinant adenovirus (Lochmüller et al., 1994; Imler et al., 1996).This is caused by homologous recombination between overlapping sequencesfrom the recombinant vector and the adenovirus constructs present in thecomplementing cell line, such as the 293 cells (Graham et al., 1977).RCA is undesirable in batches to be used in clinical trials becauseRCA 1) will replicate in an uncontrolled fashion, 2) can complementreplication-defective recombinant adenovirus, causing uncontrolledmultiplication of the recombinant adenovirus, and 3) batches containingRCA induce significant tissue damage and hence strong pathological sideeffects (Lochmüller et al., 1994). Therefore, batches to be used inclinical trials should be proven free of RCA (Ostrove, 1994).

It was generally thought that El-deleted vectors would not express anyother adenovirus genes. However, recently it has been demonstrated thatsome cell types are able to express adenovirus genes in the absence ofE1 sequences. This indicates that some cell types possess the machineryto drive transcription of adenovirus genes. In particular, it wasdemonstrated that such cells synthesize E2A and late adenovirusproteins.

In a gene therapy setting, this means that transfer of the therapeuticrecombinant gene to somatic cells not only results in expression of thetherapeutic protein but may also result in the synthesis of viralproteins. Cells that express adenoviral proteins are recognized andkilled by Cytotoxic T Lymphocytes, which thus 1) eradicates thetransduced cells and 2) causes inflammations (Bout et al., 1994a;Engelhardt et al., 1993; Simon et al., 1993). As this adverse reactionhampers gene therapy, several solutions to this problem have beensuggested, such as 1) using immunosuppressive agents after treatment, 2)retention of the adenovirus E3 region in the recombinant vector (seeEuropean patent application EP 95202213), and 3) usingtemperature-sensitive (“ts”) mutants of human adenovirus, which have apoint mutation in the E2A region rendering them temperature sensitive,as has been claimed in patent WO/28938.

However, these strategies to circumvent the immune response have theirlimitations. The use of ts mutant recombinant adenovirus diminishes theimmune response to some extent but was less effective in preventingpathological responses in the lungs (Engelhardt et al., 1994a).

The E2A protein may induce an immune response by itself, and it plays apivotal role in the switch to the synthesis of late adenovirus proteins.Therefore, it is attractive to make temperature-sensitive recombinanthuman adenoviruses.

A major drawback of this system is the fact that although the E2 proteinis unstable at the non-permissive temperature, the immunogenic proteinis still being synthesized. In addition, it is to be expected that theunstable protein does activate late gene expression, albeit to a lowextent. ts125 mutant recombinant adenoviruses have been tested, andprolonged recombinant gene expression was reported (Yang et al., 1994b;Engelhardt et al., 1994a; Engelhardt et al., 1994b; Yang et al., 1995).However, pathology in the lungs of cotton rats was still high(Engelhardt et al., 1994a), indicating that the use of ts mutantsresults in only a partial improvement in recombinant adenovirustechnology. Others (Fang et al., 1996) did not observe prolonged geneexpression in mice and dogs using ts125 recombinant adenovirus. Anadditional difficulty associated with the use of ts125 mutantadenoviruses is that a high frequency of reversion is observed. Theserevertants are either real revertants or the result of second sitemutations (Kruijer et al., 1983; Nicolas et al., 1981). Both types ofrevertants have an E2A protein that functions at normal temperature and,therefore, have toxicity similar to the wild-type virus.

BRIEF SUMMARY OF THE INVENTION

In one aspect of the invention, this problem in virus production issolved in that we have developed packaging cells that have nooverlapping sequences with a new basic vector and thus are suited forsafe large scale production of recombinant adenoviruses. One of theadditional problems associated with the use of recombinant adenovirusvectors is the host-defense reaction against treatment with adenovirus.

Briefly, recombinant adenoviruses are deleted for the E1 region. Theadenovirus E1 products trigger the transcription of the other earlygenes (E2, E3, E4), which consequently activate expression of the latevirus genes.

In another aspect of the present invention, we therefore delete E2Acoding sequences from the recombinant adenovirus genome and transfectthese E2A sequences into the (packaging) cell lines containing E1sequences to complement recombinant adenovirus vectors.

Major hurdles in this approach are 1) that E2A should be expressed tovery high levels and 2) that E2A protein is very toxic to cells.

The current invention, in yet another aspect, therefore discloses use ofthe ts125 mutant E2A gene, which produces a protein that is not able tobind DNA sequences at the non-permissive temperature. High levels ofthis protein may be maintained in the cells (because it is non-toxic atthis temperature) until the switch to the permissive temperature ismade. This can be combined with placing the mutant E2A gene under thedirection of an inducible promoter, such as, for instance, tet,methallothionein, steroid inducible promoter, retinoic acid β-receptor,or other inducible systems. However, in yet another aspect of theinvention, the use of an inducible promoter to control the moment ofproduction of toxic wild-type E2A is disclosed.

Two salient additional advantages of E2A-deleted recombinant adenovirusare 1) the increased capacity to harbor heterologous sequences and 2)the permanent selection for cells that express the mutant E2A. Thissecond advantage relates to the high frequency of reversion of ts125mutation: when reversion occurs in a cell line harboring ts125 E2A, thiswill be lethal to the cell. Therefore, there is a permanent selectionfor those cells that express the ts125 mutant E2A protein. In addition,as we in one aspect of the invention generate E2A-deleted recombinantadenovirus, we will not have the problem of reversion in ouradenoviruses.

In yet another aspect of the invention, as a further improvement, theuse of non-human cell lines as packaging cell lines is disclosed.

For GMP production of clinical batches of recombinant viruses, it isdesirable to use a cell line that has been used widely for production ofother biotechnology products. Most of the latter cell lines are ofmonkey origin, which have been used to produce, for example, vaccines.

These cells can not be used directly for the production of recombinanthuman adenovirus, as human adenovirus cannot replicate or replicatesonly to low levels in cells of monkey origin. A block in the switch ofearly to late phase of adenovirus lytic cycle underlies defectivereplication. However, host range mutations in the human adenovirusgenome are described (hr 400-404), which allow replication of humanviruses in monkey cells. These mutations reside in the gene encoding E2Aprotein (Klessig and Grodzicker, 1979; Klessig et al., 1984; Rice andKlessig, 1985; Klessig et al., 1984). Moreover, mutant viruses have beendescribed that harbor both the hr and temperature-sensitive ts125phenotype (Brough et al., 1985; Rice and Klessig, 1985).

We, therefore, generate packaging cell lines of monkey origin (e.g.,VERO, CV1) that harbor:

-   -   1) E1 sequences, to allow replication of E1/E2-defective        adenoviruses, and    -   2) E2A sequences, containing the hr mutation and the ts125        mutation, named ts400 (Brough et al., 1985; Rice and        Klessig, 1985) to prevent cell death by E2A overexpression,        and/or    -   3) E2A sequences, just containing the hr mutation, under the        control of an inducible promoter, and/or    -   4) E2A sequences, containing the hr mutation and the ts125        mutation (ts400), under the control of an inducible promoter.

Furthermore we disclose the construction of novel and improvedcombinations of packaging cell lines and recombinant adenovirus vectors.We provide:

-   -   1) A novel packaging cell line derived from diploid human        embryonic retinoblasts (“HER”) that harbors nt. 80-5788 of the        Ad5 genome. This cell line, named 911, deposited under no.        95062101 at the ECACC™, has many characteristics that make it        superior to the commonly used 293 cells (Fallaux et al., 1996);    -   2) Novel packaging cell lines that express just E1A genes and        not E1B genes. Established cell lines (and not human diploid        cells of which 293 and 911 cells are derived) are able to        express E1A to high levels without undergoing apoptotic cell        death, as occurs in human diploid cells that express E1A in the        absence of E1B. Such cell lines are able to trans-complement        E1B-defective recombinant adenoviruses, because viruses mutated        for E1B 21 kDa protein are able to complete viral replication        even faster than wild-type adenoviruses (Telling et al., 1994).        The constructs are described in detail below and are graphically        represented in FIGS. 1-5. The constructs are transfected into        the different established cell lines and are selected for high        expression of E1A. This is done by operatively linking a        selectable marker gene (e.g., NEO gene) directly to the E1B        promoter. The E1B promoter is transcriptionally activated by the        E1A gene product, and, therefore, resistance to the selective        agent (e.g., G418 in the case of NEO is used as the selection        marker) results in direct selection for desired expression of        the E1A gene;    -   3) Packaging constructs that are mutated or deleted for E1B 21        kDa, but just express the 55 kDa protein;    -   4) Packaging constructs to be used for generation of        complementing packaging cell lines from diploid cells (not        exclusively of human origin) without the need for selection with        marker genes. These cells are immortalized by expression of E1A.        However, in this particular case, expression of E1B is essential        to prevent apoptosis induced by E1A proteins. Selection of        E1-expressing cells is achieved by selection for focus formation        (immortalization), as described for 293 cells (Graham et        al., 1977) and 911 cells (Fallaux et al., 1996), that are        E1-transformed human embryonic kidney (“HEK”) cells and human        embryonic retinoblasts (“HER”), respectively;    -   5) After transfection of HER cells with construct pIG.E1B (see        FIG. 4), seven independent cell lines could be established.        These cell lines were designated PER.C1, PER.C3, PER.C4, PER.C5,        PER.C6™, PER.C8, and PER.C9. PER denotes PGK-E1-Retinoblasts.        These cell lines express E1A and E1B proteins, are stable (e.g.,        PER.C6™ for more than 57 passages), and complement E1-defective        adenovirus vectors. Yields of recombinant adenovirus obtained on        PER cells are a little higher than obtained on 293 cells. One of        these cell lines (PER.C6™) has been deposited at the ECACC™        under number 96022940;    -   6) New adenovirus vectors with extended E1 deletions (deletion        nt. 459-3510). Those viral vectors lack sequences homologous to        E1 sequences in said packaging cell lines. These adenoviral        vectors contain pIX promoter sequences and the pIX gene, as pIX        (from its natural promoter sequences) can only be expressed from        the vector and not by packaging cells (Matsui et al., 1986,        Hoeben and Fallaux, personal communication; Imler et al., 1996);    -   7) E2A-expressing packaging cell lines preferably based on        either E1A-expressing established cell lines or        E1A−E1B-expressing diploid cells (see under 2-4). E2A expression        is either under the control of an inducible promoter or the E2A        ts125 mutant is driven by either an inducible or a constitutive        promoter.    -   8) Recombinant adenovirus vectors as described before (see 6)        but carrying an additional deletion of E2A sequences;    -   9) Adenovirus packaging cells from monkey origin that are able        to trans-complement E1-defective recombinant adenoviruses. They        are preferably co-transfected with pIG.E1A.E1B and pIG.NEO and        selected for NEO resistance. Such cells expressing E1A and E1B        are able to transcomplement E1-defective recombinant human        adenoviruses but will do so inefficiently because of a block of        the synthesis of late adenovirus proteins in cells of monkey        origin (Klessig and Grodzicker, 1979). To overcome this problem,        we generate recombinant adenoviruses that harbor a host-range        mutation in the E2A gene, allowing human adenoviruses to        replicate in monkey cells. Such viruses are generated as        described in FIG. 12, except DNA from an hr-mutant is used for        homologous recombination; and    -   10) Adenovirus packaging cells from monkey origin as described        under 9, except that they will also be co-transfected with E2A        sequences harboring the hr mutation. This allows replication of        human adenoviruses lacking E1 and E2A (see under 8). E2A in        these cell lines is either under the control of an inducible        promoter or the tsE2A mutant is used. In the latter case, the        E2A gene will thus carry both the ts mutation and the hr        mutation (derived from ts400). Replication-competent human        adenoviruses have been described that harbor both mutations        (Brough et al., 1985; Rice and Klessig, 1985).

A further aspect of the invention provides otherwise improved adenovirusvectors, as well as novel strategies for generation and application ofsuch vectors and a method for the intracellular amplification of linearDNA fragments in mammalian cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures and drawings may help to understand the invention:

FIG. 1 illustrates the construction of pBS.PGK.PCRI;

FIG. 2 illustrates the construction of pIG.E1A.E1B.X;

FIGS. 3A and 3B illustrate the construction of pIG.E1A.NEO;

FIG. 4 illustrates the construction of pIG.E1A.E1B;

FIG. 5 illustrates the construction of pIG.NEO;

FIG. 6 illustrates the transformation of primary baby rat kidney (“BRK”)cells by adenovirus packaging constructs;

FIG. 7 illustrates a Western blot analysis of A549 clones transfectedwith pIG.E1 A.NEO and HER cells transfected with pIG.E1 A.E1B (PERclones);

FIG. 8 illustrates a Southern blot analysis of 293, 911 and PER celllines. Cellular DNA was extracted, HindIII digested, electrophoresed andtransferred to Hybond N+membranes (Amersham);

FIG. 9 illustrates the transfection efficiency of PER.C3, PER.C5,PER.C6T and 911 cells;

FIG. 10 illustrates construction of adenovirus vector pMLPI.TK. pMLPI.TKdesigned to have no sequence overlap with the packaging constructpIG.E1A.E1B;

FIGS. 11A and 11B illustrate new adenovirus packaging constructs whichdo not have sequence overlap with new adenovirus vectors;

FIG. 12 illustrate the generation of recombinant adenovirusIG.Ad.MLPI.TK;

FIG. 13 illustrates the adenovirus double-stranded DNA genome indicatingthe approximate locations of E1, E2, E3, E4, and L regions;

FIG. 14 illustrates the adenovirus genome is shown in the top left withthe origins of replication located within the left and right ITRs at thegenome ends.

FIG. 15 illustrates a potential hairpin conformation of asingle-stranded DNA molecule that contains the HP/asp sequence (SEQ IDNO:22 of the incorporated hereby SEQUENCE LISTING);

FIG. 16 illustrates a diagram of pICLhac;

FIG. 17 illustrates a diagram of pICLhaw;

FIG. 18 illustrates a schematic representation of pICLI;

FIG. 19 is a diagram of pICL (SEQ ID NO:21); and

FIGS. 20A-20F recite the nucleotide sequence of pICL 5620BPS DNA(circular) (SEQ ID NO:21).

DETAILED DESCRIPTION OF THE INVENTION

The so-called “minimal” adenovirus vectors according to the presentinvention retain at least a portion of the viral genome that is requiredfor encapsidation of the genome into virus particles (the encapsidationsignal), as well as at least one copy of at least a functional part or aderivative of the ITR, that is DNA sequences derived from the termini ofthe linear adenovirus genome. The vectors according to the presentinvention will also contain a transgene linked to a promoter sequence togovern expression of the transgene. Packaging of the so-called minimaladenovirus vector can be achieved by co-infection with a helper virusor, alternatively, with a packaging deficient replicating helper system,as described below.

Adenovirus-derived DNA fragments that can replicate in suitable celllines and that may serve as a packaging deficient replicating helpersystem are generated as follows. These DNA fragments retain at least aportion of the transcribed region of the “late” transcription unit ofthe adenovirus genome and carry deletions in at least a portion of theE1 region and deletions in at least a portion of the encapsidationsignal. In addition, these DNA fragments contain at least one copy of anITR. At one terminus of the transfected DNA molecule an ITR is located.The other end may contain an ITR, or alternatively, a DNA sequence thatis complementary to a portion of the same strand of the DNA moleculeother than the ITR. If, in the latter case, the two complementarysequences anneal, the free 3′-hydroxyl group of the 3′ terminalnucleotide of the hairpin-structure can serve as a primer for DNAsynthesis by cellular and/or adenovirus-encoded DNA polymerases,resulting in conversion into a double-stranded form of at least aportion of the DNA molecule. Further replication initiating at the ITRwill result in a linear double-stranded DNA molecule that is flanked bytwo ITRs and is larger than the original transfected DNA molecule (seeFIG. 13). This molecule can replicate itself in the transfected cell byvirtue of the adenovirus proteins encoded by the DNA molecule and theadenoviral and cellular proteins encoded by genes in the host-cellgenome. This DNA molecule can not be encapsidated due to its large size(greater than 39,000 base pairs) or due to the absence of a functionalencapsidation signal. This DNA molecule is intended to serve as a helperfor the production of defective adenovirus vectors in suitable celllines.

The invention also comprises a method for amplifying linear DNAfragments of variable size in suitable mammalian cells. These DNAfragments contain at least one copy of the ITR at one of the termini ofthe fragment. The other end may contain an ITR, or alternatively, a DNAsequence that is complementary to a portion of the same strand of theDNA molecule other than the ITR. If, in the latter case, the twocomplementary sequences anneal, the free 3′-hydroxyl group of the 3′terminal nucleotide of the hairpin structure can serve as a primer forDNA synthesis by cellular and/or adenovirus-encoded DNA polymerases,resulting in conversion of the displaced strand into a double strandedform of at least a portion of the DNA molecule. Further replicationinitiating at the ITR will result in a linear double-stranded DNAmolecule that is flanked by two ITRs, which is larger than the originaltransfected DNA molecule. A DNA molecule that contains ITR sequences atboth ends can replicate itself in transfected cells by virtue of thepresence of at least the adenovirus E2 proteins (viz. the DNA-bindingprotein (“DBP”), the adenovirus DNA polymerase (“Ad-pol”), and thepre-terminal protein (“pTP”). The required proteins may be expressedfrom adenovirus genes on the DNA molecule itself, from adenovirus E2genes integrated in the host-cell genome, or from a replicating helperfragment, as described above.

Several groups have shown that the presence of ITR sequences at the endof DNA molecules are sufficient to generate adenovirus minichromosomesthat can replicate, if the adenovirus-proteins required for replicationare provided in trans, for example, by infection with a helper virus (Huet al., 1992; Wang and Pearson, 1985; Hay et al., 1984). Hu et al.(1992) observed the presence and replication of symmetrical adenovirusminichromosome dimers after transfection of plasmids containing a singleITR. The authors were able to demonstrate that these dimericminichromosomes arise after tail-to-tail ligation of the single ITR DNAmolecules. In DNA extracted from defective adenovirus type 2 particles,dimeric molecules of various sizes have also been observed usingelectron-microscopy (Daniell, 1976). It was suggested that theincomplete genomes were formed by illegitimate recombination betweendifferent molecules and that variations in the position of the sequenceat which the illegitimate base pairing occurred were responsible for theheterogeneous nature of the incomplete genomes. Based on this mechanismit was speculated that, in theory, defective molecules with a totallength of up to two times the normal genome could be generated. Suchmolecules could contain duplicated sequences from either end of thegenome. However, no DNA molecules larger than the full-length virus werefound packaged in the defective particles (Daniell, 1976). This can beexplained by the size limitations that apply to the packaging. Inaddition, it was observed that in the virus particles, DNA moleculeswith a duplicated left-end predominated over those containing theright-end terminus (Daniell, 1976). This is fully explained by thepresence of the encapsidation signal near that left-end of the genome(Gräble and Hearing, 1990; Gräble and Hearing, 1992; Hearing et al.,1987).

The major problems associated with the current adenovirus-derivedvectors are:

-   -   1) The strong immunogenicity of the virus particle;    -   2) The expression of adenovirus genes that reside in the        adenoviral vectors, resulting in a Cytotoxic T-cell response        against the transduced cells; and    -   3) The low amount of heterologous sequences that can be        accommodated in the current vectors (up to maximally        approximately 8000 base pairs (“bp”) of heterologous DNA).

The strong immunogenicity of the adenovirus particle results in animmunological response of the host, even after a single administrationof the adenoviral vector. As a result of the development of neutralizingantibodies, a subsequent administration of the virus will be lesseffective or even completely ineffective. However, a prolonged orpersistent expression of the transferred genes will reduce the number ofadministrations required and may bypass the problem.

With regard to problem 2), experiments performed by Wilson andcollaborators have demonstrated that after adenovirus-mediated genetransfer into immunocompetent animals, the expression of the transgenegradually decreases and disappears approximately two to four weekspost-infection (Yang et al. 1994a; Yang et al., 1994b). This is causedby the development of a Cytotoxic T-Cell (“CTL”) response against thetransduced cells. The CTLs were directed against adenovirus proteinsexpressed by the viral vectors. In the transduced cells, synthesis ofthe adenovirus DNA-binding protein (the E2A-gene product), penton, andfiber proteins (late-gene products) could be established. Theseadenovirus proteins, encoded by the viral vector, were expressed despitedeletion of the E1 region. This demonstrates that deletion of the E1region is not sufficient to completely prevent expression of the viralgenes (Engelhardt et al., 1994a).

With regard to problem 3), studies by Graham and collaborators havedemonstrated that adenoviruses are capable of encapsidating DNA of up to105% of the normal genome size (Bett et al., 1993). Larger genomes tendto be unstable, resulting in loss of DNA sequences during propagation ofthe virus. Combining deletions in the E1 and E3 regions of the viralgenomes increases the maximum size of the foreign DNA that can beencapsidated to approximately 8.3 kb. In addition, some sequences of theE4 region appear to be dispensable for virus growth (adding another 1.8kb to the maximum encapsidation capacity). Also, the E2A region can bedeleted from the vector when the E2A gene product is provided in transin the encapsidation cell line, adding another 1.6 kb. It is, however,unlikely that the maximum capacity of foreign DNA can be significantlyincreased further than 12 kb.

We developed a new strategy for the generation and production ofhelper-free-stocks of recombinant adenovirus vectors that canaccommodate up to 38 kb of foreign DNA. Only two functional ITRsequences and sequences that can function as an encapsidation signalneed to be part of the vector genome. Such vectors are called “minimaladenovectors.” The helper functions for the minimal adenovectors areprovided in trans by encapsidation-defective replication-competent DNAmolecules that contain all the viral genes encoding the required geneproducts, with the exception of those genes that are present in thehost-cell genome, or genes that reside in the vector genome.

The applications of the disclosed inventions are outlined below and willbe illustrated in the experimental part, which is only intended for thatpurpose and should not be used to reduce the scope of the presentinvention as understood by those skilled in the art.

Use of the IG Packaging Constructs Diploid Cells

The constructs, in particular pIG.E1A.E1B, will be used to transfectdiploid human cells, such as HER, HEK, and Human Embryonic Lung cells(“HEL”). Transfected cells will be selected for transformed phenotype(focus formation) and tested for their ability to support propagation ofE1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. Such celllines will be used for the generation and (large-scale) production ofE1-deleted recombinant adenoviruses. Such cells, infected withrecombinant adenovirus, are also intended to be used in vivo as a localproducer of recombinant adenovirus, for example, for the treatment ofsolid tumors.

911 cells are used for the titration, generation, and production ofrecombinant adenovirus vectors (Fallaux et al., 1996).

HER cells transfected with pIG.E1A.E1B have resulted in sevenindependent clones (called PER cells). These clones are used for theproduction of E1-deleted (including non-overlapping adenovirus vectors)or E1-defective recombinant adenovirus vectors and provide the basis forintroduction of, for example, E2B or E2A constructs (e.g., ts125E2A, seebelow), E4 etc., that will allow propagation of adenovirus vectors thathave mutations in, for example, E2A or E4.

In addition, diploid cells of other species that are permissive forhuman adenovirus, such as the cotton rat (Sigmodon hispidus) (Pacini etal., 1984), Syrian hamster (Morin et al., 1987), or chimpanzee (Levreroet al., 1991), will be immortalized with these constructs. Such cellsinfected with recombinant adenovirus are also intended to be used invivo for the local production of recombinant adenovirus, for example,for the treatment of solid tumors.

Established Cells

The constructs, in particular pIG.E1A.NEO, can be used to transfectestablished cells, for example, A549 (human bronchial carcinoma), KB(oral carcinoma), MRC-5 (human diploid lung cell line), or GLC celllines (small cell lung cancer) (de Leij et al., 1985; Postmus et al.,1988) and selected for NEO resistance. Individual colonies of resistantcells are isolated and tested for their capacity to support propagationof E1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. Whenpropagation of E1-deleted viruses on E1A containing cells is possible,such cells can be used for the generation and production of E1-deletedrecombinant adenovirus. They can also be used for the propagation ofE1A-deleted/E1B-retained recombinant adenovirus.

Established cells can also be co-transfected with pIG.E1A.E1B andpIG.NEO (or another NEO containing expression vector). Clones resistantto G418 are tested for their ability to support propagation ofE1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK, and used forthe generation and production of E1-deleted recombinant adenovirus andwill be applied in vivo for local production of recombinant virus, asdescribed for the diploid cells (see previous discussion). All celllines, including transformed diploid cell lines or NEO-resistantestablished lines, can be used as the basis for the generation of “nextgeneration” packaging cell lines that support propagation ofE1-defective recombinant adenoviruses and that also carry deletions inother genes, such as E2A and E4. Moreover, they will provide the basisfor the generation of minimal adenovirus vectors as disclosed herein.

E2-Expressing Cell Lines

Packaging cells expressing E2A sequences are and will be used for thegeneration and large scale production of E2A-deleted recombinantadenovirus.

The newly generated human adenovirus packaging cell lines or cell linesderived from species permissive for human adenovirus (E2A or ts125E2A:E1A+E2A; E1A+E1B+E2A; E1A−E2A/ts125; E1A+E1B−E2A/ts125) ornon-permissive cell lines such as monkey cells (hrE2A or hr+ts125E2A;E1A+hrE2A; E1A+E1B+hrE2A; E1A+hrE2A/ts125; E1A−E1B+hrE2A/ts125) are andwill be used for the generation and large scale production ofE2A-deleted recombinant adenovirus vectors. In addition, they will beapplied in vivo for local production of recombinant virus, as describedfor the diploid cells (see previous discussion).

Novel Adenovirus Vectors

The newly developed adenovirus vectors harboring an E1 deletion of nt.459-3510 will be used for gene transfer purposes. These vectors are alsothe basis for the development of further deleted adenovirus vectors thatare mutated for, for example, E2A, E2B or E4. Such vectors will begenerated, for example, on the newly developed packaging cell linesdescribed above.

Minimal Adenovirus Packaging System

We disclose adenovirus packaging constructs (to be used for thepackaging of minimal adenovirus vectors) which have the followingcharacteristics:

-   -   1) the packaging construct replicates;    -   2) the packaging construct can not be packaged because the        packaging signal is deleted;    -   3) the packaging construct contains an internal hairpin-forming        sequence (see FIG. 15);    -   4) because of the internal hairpin structure, the packaging        construct is duplicated, that is, the DNA of the packaging        construct becomes twice as long as it was before transfection        into the packaging cell (in our sample, it duplicates from 35 kb        to 70 kb). This duplication also prevents packaging. Note that        this duplicated DNA molecule has ITRs at both termini (see,        e.g., FIG. 13);    -   5) this duplicated packaging molecule is able to replicate like        a “normal adenovirus” DNA molecule;    -   6) the duplication of the genome is a prerequisite for the        production of sufficient levels of adenovirus proteins required        to package the minimal adenovirus vector; and    -   7) the packaging construct has no overlapping sequences with the        minimal vector or cellular sequences that may lead to generation        of RCA by homologous recombination.

This packaging system is used to produce minimal adenovirus vectors. Theadvantages of minimal adenovirus vectors, for example, for gene therapyof vaccination purposes, are well known (accommodation of up to 38 kb;gutting of potentially toxic and immunogenic adenovirus genes).

Adenovirus vectors containing mutations in essential genes (includingminimal adenovirus vectors) can also be propagated using this system.

Use of Intracellular E2-Expressing Vectors

Minimal adenovirus vectors are generated using the helper functionsprovided in trans by packaging-deficient replicating helper molecules.The adenovirus-derived ITR sequences serve as origins of DNA replicationin the presence of at least the E2 gene products. When the E2 geneproducts are expressed from genes in the vector genome (the gene(s) mustbe driven by an E1-independent promoter), the vector genome canreplicate in the target cells. This will allow a significantly increasednumber of template molecules in the target cells, and, as a result, anincreased expression of the genes of interest encoded by the vector.This is of particular interest for application of gene therapy in cancertreatment.

Applications of Intracellular Amplification of Linear DNA Fragments

A similar approach could also be taken if amplification of linear DNAfragments is desired. DNA fragments of known or unknown sequence couldbe amplified in cells containing the E2 gene products if at least oneITR sequence is located near or at its terminus. There are no apparentconstraints on the size of the fragment. Even fragments much larger thanthe adenovirus genome (36 kb) should be amplified using this approach.It is thus possible to clone large fragments in mammalian cells withouteither shuttling the fragment into bacteria (such as E. coli) or usingthe polymerase chain reaction (“PCR”). At the end stage of a productiveadenovirus infection, a single cell can contain over 100,000 copies ofthe viral genome. In the optimal situation, the Linear DNA fragments canbe amplified to similar levels. Thus, one should be able to extract morethan 5 μg of DNA fragment per 10 million cells (for a 35-kbp fragment).This system can be used to express heterologous proteins equivalent tothe Simian Virus 40-based COS cell system for research or fortherapeutic purposes. In addition, the system can be used to identifygenes in large fragments of DNA. Random DNA fragments may be amplified(after addition of ITRs) and expressed during intracellularamplification. Election or selection of those cells with the desiredphenotype can be used to enrich the fragment of interest and to isolatethe gene.

Experiments

Generation of Cell Lines able to Transcomplement E1-DefectiveRecombinant Adenovirus Vectors 911 Cell Line

We have generated a cell line that harbors E1 sequences of adenovirustype 5 (“Ad5”), able to trans-complement E1-deleted recombinantadenovirus (Fallaux et al., 1996). This cell line was obtained bytransfection of human diploid human embryonic retinoblasts (“HER”) withpAd5XhoIC, which contains nt. 80-5788 of Ad5. One of the resultingtransformants was designated 911. This cell line has been shown to bevery useful in the propagation of E1-defective recombinant adenovirus.It was found to be superior to 293 cells. Unlike 293 cells, 911 cellslack a fully transformed phenotype, which most likely is the cause ofits better performance as an adenovirus packaging line:

-   -   1) plaque assays can be performed faster (four to five days        instead of eight to fourteen days, as on 293),    -   2) monolayers of 911 cells survive better under agar overlay, as        required for plaque assays, and    -   3) higher amplification of E1-deleted vectors is obtained.

In addition, unlike 293 cells that were transfected with shearedadenoviral DNA, 911 cells were transfected using a defined construct.Transfection efficiencies of 911 cells are comparable to those of 293.

New Packaging Constructs. Source of Adenovirus Sequences

Adenovirus sequences are derived either from pAd5.SalB, containing nt.80-9460 of human adenovirus type 5 (Bernards et al., 1983), or fromwild-type Ad5 DNA. pAd5.SalB was digested with SalI and XhoI, the largefragment was relegated, and this new clone was named pAd5.X/S. The pTNconstruct (constructed by Dr. R. Vogels, IntroGene, Leiden, TheNetherlands) was used as a source for the human PGK promoter and the NEOgene.

Human PGK Promoter and NEO^(R) Gene

Transcription of E1A sequences in the new packaging constructs is drivenby the human PGK promoter (Michelson et al., 1983; Singer-Sam et al.,1984), derived from plasmid pTN (gift of R. Vogels), which uses pUCI 19(Vieira and Messing, 1987) as a backbone. This plasmid was also used asa source for NEO gene fused to the Hepatitis B Virus (“HBV”)poly-adenylation signal.

Fusion of PGK Promoter to E1 Genes

As shown in FIG. 1, in order to replace the E1 sequences of Ad5 (ITR,origin of replication, and packaging signal) by heterologous sequenceswe have amplified E1 sequences (nt. 459 to nt. 960) of Ad5 by PCR, usingprimers Ea-l (SEQ ID NO: 1) and Ea-2 (SEQ ID NO:2) (see Table I). Theresulting PCR product was digested with ClaI and ligated into Bluescript(Stratagene), predigested with ClaI and EcoRV, resulting in constructpBS.PCR1. TABLE I Primer Sequences. Name (SEQ ID NO) Sequence FunctionPrimer Ea-1 CGTGTAGTGT ATTTATACCCG PCR amplification (SEQ ID NO:1) a AdSnt 459 −> Primer Ea-2 TCGTCACTGG GTGGAAACCCA PCR amplification (SEQ IDNO:2) a Ad5 nt 960 <− Primer Ea-3 TACCCGCCGT CCTAAAATGGC nt 1284-1304 ofAd5 (SEQ ID NO:3) a genome Primer Ea-5 TGGACTTGAG CTGTAAACGC nt1514-1533 of Ad5 (SEQ ID NO:4) a genome Primer Ep-2GCCTCCATGG AGGTCAGATGT nt 1721-1702 of Ad5: (SEQ ID NO:5) a introductionof NcoI site Primer Eb-1 GCTTGAGCCC GAGACATGTC nt 3269-3289 of Ad5 (SEQID NO:6) a genome Primer Eb-2 CCCCTCGAGC TCAATCTGTATCTT nt 3508-3496 ofAd5 (SEQ ID NO:7) a genome: introduction of XhoI site Primer SV40-1GGGGGATCCG AACTTGTTTA Introduction BamHI (SEQ ID NO:8) a TTGCAGC site(nt 2182-2199 of pMLP.TK) adaption of recombinant adenoviruses PrimerSV40-2 GGGAGATCTA GACATGATAA Introduction Bg1II (SEQ ID NO:9) a GATACsite (nt 2312-2297 of pMLP.TK) Primer Ad5-1 GGGAGATCTG TACTGAAATGIntroduction of (SEQ ID NO:10) a TGTGGGC Bg1II site (nt 2496-2514 ofpMLP.TK) Primer Ad5-2 GGAGGCTGCA GTCTCCAACGGCGT Rnt 2779-2756 of (SEQ IDNO:11) a PMLP.TK Primer ITR1 GGGGGATCCT CAAATCGTCA nt 35737-35757 of(SEQ ID NO:12) a CTTCCGT Ad5 (introduction of BamHI site) Primer ITR2GGGGTCTAGA CATCATCAAT nt 35935-35919 of (SEQ ID NO:13) a AATATAC Ad5(introduction of XbaI site) PCR primer PCR/MLP1 GGCGAATTCG TCGACATCAT(Ad5 nt. 10-18) (SEQ ID NO:14) b CAATAATATA CC PCT primer PCR/MLP2GGCGAATTCG GTACCATCAT (Ad5 nt. 10-18) (SEQ ID NO:15) b CAATAATATA CC PCTprimer PCR/MLP3 CTGTGTACAC CGGCGCA (Ad5 nt. 200-184) (SEQ ID NO:16) bPCT primer HP/asp1 5′-GTACACTGAC CTAGTGCCGC (SEQ ID NO:17) C CCGGGCAAAGCCCGGGCGGC ACTAGGTCAG PCT primer HP/asp2 5′-GTACCTGACC TAGTGCCGCC (SEQID NO:18) c CGGGCTTTGC CCGGGCGGCA CTAGGTCAGT PCT primer HP/cla15′-GTACATTGAC CTAGTGCCGC (SEQ ID NO:19) d CCGGGCAAAG CCCGGGCGGCACTAGGTCAA TCGAT PCT primer HP/cla2 5′-GTACATCGAT TGACCTAGTG (SEQ IDNO:20) d CCGCCCGGGC TTTGCCCGGG CGGCACTAGG TCAATa-Primers used for PCR amplification of DNA fragments used forgeneration of constructs described in this patent application.b-PCR primers sets to be used to create the SalI and Asp718 sitesjuxtaposed to the ITR sequences.c-Synthetic oligonucleotide pair used to generate a synthetic hairpin,recreates an Asp718 site at one of the termini if inserted in Asp718site.d-Synthetic oligonucleotide pair used to generate a synthetic hairpin,contains the ClaI recognition site to be used for hairpin formation.

Vector pTN was digested with restriction enzymes EcoRI (partially) andScaI and the DNA fragment containing the PGK promoter sequences wasligated into PBS.PCRI digested with ScaI and EcoRI. The resultingconstruct pBS.PGK.PCR1 contains the human PGK promoter operativelylinked to Ad5 E1 sequences from nt. 459 to nt. 916.

Construction of pIG.E1A.E1B.X

As shown in FIG. 2, pIG.E1A.E1B.X was made by replacing the ScaI-BspEIfragment of pAT.X/S by the corresponding fragment from pBS.PGK.PCR1(containing the PGK promoter linked to E1A sequences). pIG.E1A.E1B.Xcontains the E1A and E1B coding sequences under the direction of the PGKpromoter. As Ad5 sequences from nt. 459 to nt. 5788 are present in thisconstruct, pIX protein of adenovirus is also encoded by this plasmid.

Construction of pIG.E1A.NEO

As shown in FIG. 3A, in order to introduce the complete E1B promoter andto fuse this promoter in such a way that the AUG codon of E1B 21 kDaexactly functions as the AUG codon of NEO^(R), we amplified the E1Bpromoter using primers Ea-3 (SEQ ID NO:3) and Ep-2 (SEQ ID NO:5), whereprimer Ep-2 (SEQ ID NO:5) introduces an NcoI site in the PCR fragment.The resulting PCR fragment, named PCRII, was digested with HpaI and NcoIand ligated into pAT-X/S, which was predigested with HpaI and with NcoI.The resulting plasmid was designated pAT.X/S.PCR2. The NcoI-StuIfragment of pTN, containing the NEO gene and part of the HBVpoly-adenylation signal, was cloned into pAT.X/S.PCR2 (digested withNcoI and NruI). The resulting construct: pAT.PCR2.NEO.

As shown in FIG. 3B, the poly-adenylation signal was completed byreplacing the ScaI-SalI fragment of pAT-PCR2-NEO with the correspondingfragment of pTN (resulting in pAT.PCR2.NEO.p(A)). The ScaI-XbaI ofpAT.PCR2.NEO.p (A) was replaced by the corresponding fragment ofpIG.E1A.E1B-X, containing the PGK promoter linked to E1A genes. Theresulting construct was named pIG.E1A.NEO, and thus contains Ad5 E1sequences (nt. 459 to nt. 1713) under the control of the human PGKpromoter.

Construction of pIG.E1A.E1B

As shown in FIG. 4, pIG.E1A.E1B was made by amplifying the sequencesencoding the N-terminal amino acids of E1B 55kDa using primers Eb-1 (SEQID NO:6) and Eb-2 (SEQ ID NO:7) (introduces an XhoI site). The resultingPCR fragment was digested with BglII and cloned into BglII/NruI ofpAT-X/S, thereby obtaining pAT.PCR3.

pIG.E1A.E1B was constructed by introducing the HBV poly(A) sequences ofpIG.E1A.NEO downstream of E1B sequences of pAT.PCR3 by exchange ofXbaI-SalI fragment of pIG.E1A.NEO and the XbaI XhoI fragment ofpAT.PCR3.

pIG.E1A.E1B contains nt. 459 to nt. 3510 of Ad5, which encode the E1Aand E1B proteins. The E1B sequences are terminated at the spliceacceptor at nt. 3511. No pIx sequences are present in this construct.

Construction of pIG.NEO

As shown in FIG. 5, pIG.NEO was generated by cloning the HpaI-ScaIfragment of pIG.E1A.NEO, containing the NEO gene under the control ofthe Ad.5 E1B promoter, into pBS digested with EcoRV and ScaI.

This construct is of use when established cells are transfected withE1A.E1B constructs and NEO selection is required. Because NEO expressionis directed by the E1B promoter, NEO-resistant cells are expected toco-express E1A, which is also advantageous for maintaining high levelsof expression of E1A during long-term culture of the cells.

Testing of Constructs

The integrity of the constructs pIG.E1A.NEO, pIG.E1A.E1B.X andpIG.E1A.E1B was assessed by restriction enzyme mapping; furthermore,parts of the constructs that were obtained by PCR analysis wereconfirmed by sequence analysis. No changes in the nucleotide sequencewere found.

The constructs were transfected into primary Baby Rat Kidney (“BRK”)cells and tested for their ability to immortalize (pIG.E1A.NEC) or fullytransform (pAd5.XhoIC, pIG.E1A.E1B.X, and pIG.E1A.E1B) these cells.

Kidneys of six-day-old WAG-Rij rats were isolated, homogenized, andtrypsinized. Subconfluent dishes (diameter 5 cm) of the BRK cellcultures were transfected with 1 or 5 μg of pIG.NEO, pIG.E1A.NEO,pIG.E1A.E1B, pIG.E1A.E1B.X, pAd5XhoIC, or pIG.E1A.NEO together withPDC26 (Van der Elsen et al., 1983), carrying the Ad5.E1B gene undercontrol of the SV40 early promoter. Three weeks post-transfection, whenfoci were visible, the dishes were fixed, Giemsa stained, and the focicounted.

An overview of the generated adenovirus packaging constructs and theirability to transform BRK is presented in FIG. 6. The results indicatethat the constructs pIG.E1A.E1B and pIG.E1A.E1B.X are able to transformBRK cells in a dose-dependent manner. The efficiency of transformationis similar for both constructs and is comparable to what was found withthe construct that was used to make 911 cells, namely pAd5.XhoIC.

As expected, pIG.E1A.NEO was hardly able to immortalize BRK. However,co-transfection of an E1B expression construct (PDC26) did result in asignificant increase in the number of transformants (18 versus 1),indicating that E1A encoded by pIG.E1A.NEO is functional. We conclude,therefore, that the newly generated packaging constructs are suited forthe generation of new adenovirus packaging lines.

Generation of Cell Lines with New Packaging Constructs, Cell Lines, andCell Culture

Human A549 bronchial carcinoma cells (Shapiro et al., 1978), humanembryonic retinoblasts (“HER”), Ad5-E1-transformed human embryonickidney (“HEK”) cells (293; Graham et al., 1977), and Ad5-transformed HERcells (911; Fallaux et al., 1996)) and PER cells were grown inDulbecco's Modified Eagle Medium (“DMEM”) supplemented with 10% FetalCalf Serum (“FCS”) and antibiotics in a 5% CO₂ atmosphere at 37° C. Cellculture media, reagents, and sera were purchased from Gibco Laboratories(Grand Island, N.Y.). Culture plastics were purchased from Greiner(Nirtingen, Germany) and Corning (Corning, N.Y.).

Viruses and Virus Techniques

The construction of adenoviral vectors IG.Ad.MLP.nls.lacZ,IG.Ad.MLP.luc, IG.Ad.MLP.TK, and IG.Ad.CMV.TK is described in detail inEuropean patent application EP 95202213. The recombinant adenoviralvector IG.Ad.MLP.nls.lacZ contains the E. coli lacZ gene, encodingβ-galactosidase, under control of the Ad2 major late promoter (“MLP”).IG.Ad.MLP.luc contains the firefly luciferase gene driven by the Ad2MLP. Adenoviral vectors IG.Ad.MLP.TK and IG.Ad.CMV.TR contain the HerpesSimplex Virus thymidine kinase (“TK”) gene under the control of the Ad2MLP and the Cytomegalovirus (“CMV”) enhancer/promoter, respectively.

Transfections

All transfections were performed by calcium-phosphate precipitation DNA(Graham and Van der Eb, 1973) with the GIBCO Calcium PhosphateTransfection System (GIBCO BRL Life Technologies Inc., Gaithersburg,Md., USA), according to the manufacturer's protocol.

Western Blotting

Subconfluent cultures of exponentially growing 293, 911 andAd5-E1-transformed A549 and PER cells were washed with PBS and scrapedin Fos-RIPA buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 01%sodium dodecyl sulphate (“SDS”), 1% NA-DOC, 0.5 mM phenyl methylsulphonyl fluoride (“PMSF”), 0.5 mM trypsin inhibitor, 50 mM NaF and 1mM sodium vanadate). After ten minutes at room temperature, lysates werecleared by centrifugation. Protein concentrations were measured with theBiorad protein assay kit, and 25 μg total cellular protein was loaded ona 12.5% SDS-PAA gel. After electrophoresis, proteins were transferred tonitrocellulose (one hour at 300 mA). Prestained standards (Sigma, USA)were run in parallel. Filters were blocked with 1% bovine serum albumin(“BSA”) in TBST (10 mM Tris, pH 8.15 mM NaCl, and 0.05% TWEEN™-20) forone hour. First antibodies were the mouse monoclonal anti-Ad5-E1B-55-kDaantibody A1C6 (Zantema et al., unpublished), the rat monoclonalanti-Ad5-E1B-221-kDa antibody C1G11 (Zantema et al., 1985). The secondantibody was a horseradish peroxidase-labeled goat anti-mouse antibody(Promega). Signals were visualized by enhanced chemiluminescence(Amersham Corp, UK).

Southern Blot Analysis

High molecular weight DNA was isolated and 10 μg was digested tocompletion and fractionated on a 0.7% agarose gel. Southern blottransfer to Hybond N+(Amersham, UK) was performed with a 0.4 M NaOH, 0.6M NaCl transfer solution (Church and Gilbert, 1984). Hybridization wasperformed with a 2463-nt SspI-HindIII fragment from pAd5.SalB (Bernardset al., 1983). This fragment consists of Ad5 bp. 342-2805. The fragmentwas radiolabeled with α-³²P-dCTP with the use of random hexanucleotideprimers and Klenow DNA polymerase. The Southern blots were exposed to aKodak XAR-5 film at −80° C. and to a Phospho-Imager screen that wasanalyzed by B&L Systems' Molecular Dynamics software.

A549

Ad5-E1-transformed A549 human bronchial carcinoma cell lines weregenerated by transfection with pIG.E1A.NEO and selection for G418resistance. Thirty-one G418-resistant clones were established.Co-transfection of pIG.E1A.E1B with pIG.NEO yielded seven G418-resistantcell lines.

PER

Ad5-E1-transformed HER cells were generated by transfection of primaryHER cells with plasmid pIG.E1A.E1B. Transformed cell lines wereestablished from well-separated foci. We were able to establish sevenclonal cell lines, which we called PER.C1, PER.C3, PER.C4, PER.C5,PER.C6™, PER.C8, and PER.C9. One of the PER clones, namely PER.C6™, hasbeen deposited under the Budapest Treaty under number ECACC™ 96022940with the Centre for Applied Microbiology and Research of Porton Down,UK, on Feb. 29, 1996. In addition, PER.C6™ is commercially availablefrom IngroGene, B.V., Leiden, NL. Expression of Ad5 E1A and E1B genes intransformed A549 and PER cells

Expression of the Ad5 E1A and the 55 kDa and 21 kDa E1B proteins in theestablished A549 and PER cells was studied by means of Western blottingwith the use of monoclonal antibodies (“Mab”). Mab M73 recognizes theE1A products, whereas Mabs AIC6 and C1G11 are directed against the 55kDa and 21 kDa E1B proteins, respectively.

The antibodies did not recognize proteins in extracts from the parentalA549 or the primary HER cells (data not shown). None of the A549 clonesthat were generated by co-transfection of pIG.NEO- andpIG.E1A.E1B-expressed detectable levels of E1A or E1B proteins (notshown). Some of the A549 clones that were generated by transfection withpIG.E1A.NEO expressed the Ad5 E1A proteins (see FIG. 7), but the levelswere much lower than those detected in protein lysates from 293 cells.The steady state E1A levels detected in protein extracts from PER cellswere much higher than those detected in extracts from A549-derivedcells. All PER cell lines expressed similar levels of E1A proteins (FIG.7). The expression of the E1B proteins, particularly in the case of E1B55 kDa, was more variable. Compared to 911 and 293, the majority of thePER clones express high levels of E1B 55 kDa and 21 kDa. The steadystate level of E1B 21 kDa was the highest in PER.C3. None of the PERclones lost expression of the Ad5 E1 genes upon serial passage of thecells (not shown). We found that the level of E1 expression in PER cellsremained stable for at least 100 population doublings. We decided tocharacterize the PER clones in more detail.

Southern Analysis of PER Clones

To study the arrangement of the Ad5-E1 encoding sequences in the PERclones, we performed Southern analyses. Cellular DNA was extracted fromall PER clones and from 293 and 911 cells. The DNA was digested withHindIII, which cuts once in the AdS E1 region. Southern hybridization onHindIII-digested DNA, using a radiolabeled Ad5-E1-specific probe,revealed the presence of several integrated copies of pIG.E1A.E1B in thegenome of the PER clones. FIG. 8 shows the distribution pattern of E1sequences in the high molecular weight DNA of the different PER celllines. The copies are concentrated in a single band, which suggests thatthey are integrated as tandem repeats. In the case of PER.C3, C5, C6,and C9, we found additional hybridizing bands of low molecular weightthat indicate the presence of truncated copies of pIG.E1A.E1B. Thenumber of copies was determined with the use of a Phospho-Imager. Weestimated that PER.C1, C3, C4, C5, C6, C8, and C9 contain 2, 88, 5, 4,5, 5, and 3 copies of the Ad5 E1 coding region, respectively, and that911 and 293 cells contain 1 and 4 copies of the Ad5 E1 sequences,respectively.

Transfection Efficiency

Recombinant adenovectors are generated by co-transfection of adaptorplasmids and the large ClaI fragment of AdS into 293 cells (see Europeanpatent application EP 95202213). The recombinant virus DNA is formed byhomologous recombination between the homologous viral sequences that arepresent in the plasmid and the adenovirus DNA. The efficacy of thismethod, as well as that of alternative strategies, is highly dependenton the transfectability of the helper cells. Therefore, we compared thetransfection efficiencies of some of the PER clones with 911 cells,using the E. coli β-galactosidase-encoding lacZ gene as a reporter (seeFIG. 9).

Production of Recombinant Adenovirus

Yields of recombinant adenovirus obtained after inoculation of 293, 911,PER.C3, PER.C5, and PER.C6TM with different adenovirus vectors arepresented in Table II. The results indicate that the yields obtained onPER cells are at least as high as those obtained on the existing celllines. In addition, the yields of the novel adenovirus vectorIG.Ad.MLPI.TK are similar or higher than the yields obtained for theother viral vectors on all cell lines tested. TABLE II IG.Ad. IG.Ad.IG.Ad. Passage CMV. CMV. MLPI. Producer Cell number lacZ TK TK d1313Mean 293 6.0 5.8 24 34 17.5 911 8 14 34 180 59.5 PER.C3 17 8 11 44 4025.8 PER.C5 15 6 17 36 200 64.7 PER.C6 ™ 36 10 22 58 320 102Yields × 10⁻⁸ pfu/T175 flask.

Table II. Yields of different recombinant adenoviruses obtained afterinoculation of adenovirus E1 packaging cell lines 293, 911, PER.C3,PER.C5, and PER.C6™. The yields are the mean of two differentexperiments. IG.Ad.CMV.lacZ and IG.Ad.CMV.TK are described in Europeanpatent application EP 95202213. The construction of IG.Ad.MLPI.TK isdescribed in this patent application. Yields of virus per T80 flask weredetermined by plaque assay on 911 cells, as described [Fallaux, 1996#1493].

Generation of New Adenovirus Vectors

The used recombinant adenovirus vectors (see European patent applicationEP 95202213) are deleted for E1 sequences from nt. 459 to nt. 3328.

As construct pE1A.E1B contains Ad5 sequences nt. 459 to nt. 3510, thereis a sequence overlap of 183 nt. between E1B sequences in the packagingconstruct pIG.E1A.E1B and recombinant adenoviruses, such as, forexample, IG.Ad.MLP.TK. The overlapping sequences were deleted from thenew adenovirus vectors. In addition, non-coding sequences derived fromlacZ, which are present in the original constructs, were deleted aswell. This was achieved (see FIG. 10) by PCR amplification of the SV40poly(A) sequences from pMLP.TK using primers SV40-1 (SEQ ID NO:8)(introduces a BamHI site) and SV40-2 (SEQ ID NO:9) (introduces a BglIIsite). In addition, Ad5 sequences present in this construct wereamplified from nt. 2496 (Ad5-1 (SEQ ID NO:10), introduces a BglII site)to nt. 2779 (Ad5-2 (SEQ ID NO: 11)). Both PCR fragments were digestedwith BglII and were ligated. The ligation product was PCR amplifiedusing primers SV40-1 (SEQ ID NO:8) and Ad5-2 (SEQ ID NO:11). The PCRproduct obtained was cut with BamHI and AflII and was ligated intopMLP.TK predigested with the same enzymes. The resulting construct,named pMLPI.TK, contains a deletion in adenovirus E1 sequences from nt.459 to nt. 3510.

Packaging System

The combination of the new packaging construct pIG.E1A.E1B and therecombinant adenovirus pMLPI.TK, which do not have any sequence overlap,are presented in FIGS. 11A and 11B. In these figures, the originalsituation is also presented with the sequence overlap indicated.

The absence of overlapping sequences between pIG.E1A.E1B and pMLPI.TK(FIG. 11A) excludes the possibility of homologous recombination betweenpackaging construct and recombinant virus, and is therefore asignificant improvement for production of recombinant adenovirus ascompared to the original situation.

In FIG. 11B, the situation is depicted for pIG.E1A.NEO andIG.Ad.MLPI.TK. pIG.E1A.NEO, when transfected into established cells, isexpected to be sufficient to support propagation of E1-deletedrecombinant adenovirus. This combination does not have any sequenceoverlap, preventing generation of RCA by homologous recombination. Inaddition, this convenient packaging system allows the propagation ofrecombinant adenoviruses that are deleted just for E1A sequences and notfor E1B sequences. Recombinant adenoviruses expressing E1B in theabsence of E1A are attractive, as the E1B protein, in particular E1B 19kDa, is able to prevent infected human cells from lysis by TumorNecrosis Factor (“TNF”) (Gooding et al., 1991).

Generation of Recombinant Adenovirus Derived from pMLPI.TK

Recombinant adenovirus was generated by co-transfection of 293 cellswith SalI linearized pMLPI.TK DNA and ClaI linearized Ad5 wt DNA. Theprocedure is schematically represented in FIG. 12.

Outline of the Strategy to Generate Packaging Systems for MinimalAdenovirus Vector

Name convention of the plasmids used:

-   -   p plasmid    -   I ITR (Adenovirus Inverted Terminal Repeat)    -   C CMV Enhancer/Promoter Combination    -   L Firefly Luciferase Coding Sequence hac, haw Potential hairpin        that can be formed after digestion with restriction endonuclease        Asp718 in its correct and in the reverse orientation,        respectively (FIG. 15 (SEQ ID NO:22)).

For example, pICLhaw is a plasmid that contains the adenovirus ITRfollowed by the CMV-driven luciferase gene and the Asp718 hairpin in thereverse (non-functional) orientation.

Experiment Series 1

The following demonstrates the competence of a synthetic DNA sequencethat is capable of forming a hairpin-structure to serve as a primer forreverse strand synthesis for the generation of double-stranded DNAmolecules in cells that contain and express adenovirus genes.

Plasmids pICLhac, pICLhaw, pICLI and pICL (SEQ ID NO:21) were generatedusing standard techniques. The schematic representation of theseplasmids is shown in FIGS. 16-19.

Plasmid pICL (SEQ ID NO:21) is derived from the following plasmids:

-   -   nt. 11-457 pMLP10 (Levrero et al., 1991);    -   nt. 458-1218 pCMVβ (Clontech, EMBL Bank No. U02451);    -   nt. 1219-3016 pMLP.luc (IntroGene, Leiden, NL, unpublished); and    -   nt. 3017-5620 pBLCAT5 (Stein and Whelan, 1989).

The plasmid has been constructed as follows:

The tet gene of plasmid pMLP10 has been inactivated by deletion of theBamHI-SalI fragment to generate pMLP10ΔSB. Using primer set PCR/MLP1(SEQ ID NO:14) and PCR/MLP3 (SEQ ID NO:16), a 210 bp fragment containingthe Ad5-ITR, flanked by a synthetic SalI restriction site, was amplifiedusing pMLP10 DNA as the template. The PCR product was digested with theenzymes EcoRI and SgrAI to generate a 196 bp fragment. Plasmid pMLP10ΔSBwas digested with EcoRI and SgrAI to remove the ITR. This fragment wasreplaced by the EcoRI-SgrAI-treated PCR fragment to generate pMLP/SAL.Plasmid pCMV-Luc was digested with PvuII to completion and recirculatedto remove the SV40-derived poly-adenylation signal and Ad5 sequenceswith exception of the Ad5 left-terminus. In the resulting plasmid,pCMV-lucΔAd, the Ad5 ITR was replaced by the Sal-site-flanked ITR fromplasmid pMLP/SAL by exchanging the XmnI-SacII fragments. The resultingplasmid, pCMV-lucΔAd/SAL, the Ad5 left terminus, and the CMV-drivenluciferase gene were isolated as an SalI-SmaI fragment and inserted inthe SalI and HpaI digested plasmid pBLCATS to form plasmid pICL (SEQ IDNO:21). Plasmid pICL is represented in FIG. 19; its sequence ispresented in FIGS. 20A-20F (SEQ ID NO:21).

The plasmid pICL (SEQ ID NO:21) contains the following features:

-   -   nt. 1-457 AdS left terminus (Sequence 1-457 of human adenovirus        type 5);    -   nt. 458-969 Human cytomegalovirus enhancer and immediate early        promoter (see Boshart et al., A Very Strong Enhancer is Located        Upstream of an Immediate Early Gene of Human Cytomegalovirus”,        Cell 41, pp. 521-530 (1985), hereby incorporated herein by        reference) (from plasmid pCMVβ, Clontech, Palo Alto, USA);    -   nt. 970-1204 SV40 19S exon and truncated 16/19S intron (from        plasmid pCMVβ);    -   nt. 1218-2987 Firefly luciferase gene (from pMLP.luc);    -   nt. 3018-3131 SV40 tandem poly-adenylation signals from late        transcript, derived from plasmid pBLCAT5);    -   nt. 3132-5620 pUC12 backbone (derived from plasmid pBLCAT5); and    -   nt. 4337-5191 β-lactamase gene (Amp-resistance gene, reverse        orientation).        Plasmid pICLhac and pICLhaw

Plasmids pICLhac and pICLhaw were derived from plasmid pICL (SEQ IDNO:21) by digestion of the latter plasmid with the restriction enzymeAsp718. The linearized plasmid was treated with Calf-Intestine AlkalinePhosphatase to remove the 51 phosphate groups. The partiallycomplementary synthetic single-stranded oligonucleotide Hp/asp1 (SEQ IDNO:17) and Hp/asp2 (SEQ ID NO:18) were annealed and phosphorylated ontheir 5′ ends using T4-polynucleotide kinase.

The phosporylated double-stranded oligomers were mixed with thedephosporylated pICL fragment and ligated. Clones containing a singlecopy of the synthetic oligonucleotide inserted into the plasmid wereisolated and characterized using restriction enzyme digests. Insertionof the oligonucleotide into the Asp718 site will at one junctionrecreate an Asp718 recognition site, whereas at the other junction, therecognition site will be disrupted. The orientation and the integrity ofthe inserted oligonucleotide were verified in selected clones bysequence analyses. A clone containing the oligonucleotide in the correctorientation (the Asp718 site close to the 3205 EcoRI site) was denotedpICLhac. A clone with the oligonucleotide in the reverse orientation(the Asp718 site close to the SV40-derived poly signal) was designatedpICLhaw. Plasmids pICLhac and pICLhaw are represented in FIGS. 16 and17.

Plasmid pICLI was created from plasmid pICL (SEQ ID NO:21) by insertionof the SalI-SgrAI fragment from pICL containing the Ad5-ITR into theAsp718 site of pICL. The 194 bp SalI-SgrAI fragment was isolated frompICL (SEQ ID NO:21), and the cohesive ends were converted to blunt endsusing E. coli DNA polymerase I (Klenow fragment) and dNTPs. The Asp718cohesive ends were converted to blunt ends by treatment with mungbeannuclease. Clones were generated by ligation that contain the ITR in theAsp718 site of plasmid pICL (SEQ ID NO:21). A clone that contained theITR fragment in the correct orientation was designated pICLI (see FIG.18). Generation of adenovirus Ad-CMV-hcTK: recombinant adenovirus wasconstructed according to the method described in European patentapplication EP 95202213. Two components are required to generate arecombinant adenovirus. First, an adaptor-plasmid containing the leftterminus of the adenovirus genome containing the ITR and the packagingsignal, an expression cassette with the gene of interest, and a portionof the adenovirus genome which can be used for homologous recombination.In addition, adenovirus DNA is needed for recombination with theaforementioned adaptor plasmid. In the case of Ad-CMV-hcTK, the plasmidPCMV.TK was used as a basis. This plasmid contains nt. 1-455 of theadenovirus type 5 genome, nt. 456-1204 derived from pCMVβ (Clontech, thePstI-StuI fragment that contains the CMV enhancer promoter and the16S/19S intron from Simian Virus 40), the HSV TK gene (described inEuropean patent application EP 95202213), the SV40-derivedpolyadenylation signal (nt 2533-2668 of the SV40 sequence), followed bythe BglII-ScaI fragment of Ad5 (nt. 3328-6092 of the Ad5 sequence).These fragments are present in a pMLP10-derived (Levrero et al., 1991)backbone. To generate plasmid pAD-CMVhc-TK, plasmid pCMV.TK was digestedwith ClaI (the unique ClaI-site is located just upstream of the TK openreading frame) and dephosphorylated with Calf-Intestine AlkalinePhosphate. To generate a hairpin-structure, the syntheticoligonucleotides HP/cla1 (SEQ ID NO:19) and HP/cla2 (SEQ ID NO:20) wereannealed and phosphorylated on their 5-OH groups with T4-polynucleotidekinase and ATP. The double-stranded oligonucleotide was ligated with thelinearized vector fragment and used to transform E. coli strain “Sure”.Insertion of the oligonucleotide into the ClaI site will disrupt theClaI recognition sites. The oligonucleotide contains a new ClaI sitenear one of its termini. In selected clones, the orientation and theintegrity of the inserted oligonucleotide was verified by sequenceanalyses. A clone containing the oligonucleotide in the correctorientation (the ClaI site at the ITR side) was denoted pAd-CMV-hcTK.This plasmid was co-transfected with ClaI digested wild-type Adenovirustype 5 DNA into 911 cells. A recombinant adenovirus in which theCMV-hcTK expression cassette replaces the E1 sequences was isolated andpropagated using standard procedures.

To study whether the hairpin can be used as a primer for reverse strandsynthesis on the displaced strand after replication had started at theITR, the plasmid pICLhac is introduced into 911 cells (human embryonicretinoblasts transformed with the adenovirus E1 region). The plasmidpICLhaw serves as a control, which contains the oligonucleotide pairHP/asp1 (SEQ ID NO: 17) and HP/asp2 (SEQ ID NO: 18) in the reverseorientation but is further completely identical to plasmid pICLhac. Alsoincluded in these studies are plasmids pICLI and pICL (SEQ ID NO:21). Inthe plasmid pICLI, the hairpin is replaced by an adenovirus ITR. PlasmidpICL (SEQ ID NO:21) contains neither a hairpin nor an ITR sequence.These plasmids serve as controls to determine the efficiency ofreplication by virtue of the terminal-hairpin structure. To provide theviral products other than the E1 proteins (these are produced by the 911cells) required for DNA replication, the cultures are infected with thevirus IG.Ad.MLPI.TK after transfection. Several parameters are beingstudied to demonstrate proper replication of the transfected DNAmolecules. First, DNA extracted from the cell cultures transfected withaforementioned plasmids and infected with IG.Ad.MLPI.TK virus is beinganalyzed by Southern blotting for the presence of the expectedreplication intermediates, as well as for the presence of the duplicatedgenomes. Furthermore, virus is isolated from the transfected andIG.Ad.MLPI.TK infected cell populations that is capable of transferringand expressing a luciferase marker gene into luciferase negative cells.

Plasmid DNA of plasmids pICLhac, pICLhaw, pICLI, and pICL (SEQ ID NO:21)have been digested with restriction endonuclease SalI and treated withmungbean nuclease to remove the 4 nucleotide single-stranded extensionof the resulting DNA fragment. In this manner, a natural adenovirus 5′ITR terminus on the DNA fragment is created. Subsequently, both thepICLhac and pICLhaw plasmids were digested with restriction endonucleaseAsp718 to generate the terminus capable of forming a hairpin structure.The digested plasmids are introduced into 911 cells, using the standardcalcium phosphate co-precipitation technique, four dishes for eachplasmid. During the transfection, for each plasmid two of the culturesare infected with the IG.Ad.MLPI.TK virus using five infectiousIG.Ad.MLPI.TK particles per cell. At twenty-hours post-transfection andforty hours post-transfection, one Ad.tk-virus-infected and oneuninfected culture are used to isolate small molecular-weight DNA usingthe procedure devised by Hirt. Aliquots of isolated DNA are used forSouthern analysis. After digestion of the samples with restrictionendonuclease EcoRI using the luciferase gene as a probe, a hybridizingfragment of approximately 2.6 kb is detected only in the samples fromthe adenovirus infected cells transfected with plasmid pICLhac. The sizeof this fragment is consistent with the anticipated duplication of theluciferase marker gene. This supports the conclusion that the insertedhairpin is capable of serving as a primer for reverse strand synthesis.The hybridizing fragment is absent if the IG.Ad.MLPI.TK virus is omittedor if the hairpin oligonucleotide has been inserted in the reverseorientation.

The restriction endonuclease DpnI recognizes the tetranucleotidesequence 5′-GATC-3′ but cleaves only methylated DNA (that is, onlyplasmid DNA propagated in and derived from E. coli, not DNA that hasbeen replicated in mammalian cells). The restriction endonuclease MboIrecognizes the same sequences, but cleaves only unmethylated DNA (viz.DNA propagated in mammalian cells). DNA samples isolated from thetransfected cells are incubated with MboI and DpnI and analyzed withSouthern blots. These results demonstrate that only in the cellstransfected with the PICLhac and the pICLI plasmids are largeDpnI-resistant fragments present that are absent in the MboI treatedsamples. These data demonstrate that only after transfection of plasmidspICLI and pICLhac does replication and duplication of the fragmentsoccur.

These data demonstrate that in adenovirus-infected cells, linear DNAfragments that have on one terminus an adenovirus-derived ITR and at theother terminus a nucleotide sequence that can anneal to sequences on thesame strand when present in single-stranded form, thereby generate ahairpin structure and will be converted to structures that have invertedterminal repeat sequences on both ends. The resulting DNA.molecules willreplicate by the same mechanism as the wild-type adenovirus genomes.

Experiment Series 2

The following demonstrates that the DNA molecules that contain aluciferase marker gene, a single copy of the ITR, the encapsidationsignal, and a synthetic DNA sequence that is capable of forming ahairpin structure are sufficient to generate DNA molecules that can beencapsidated into virions.

To demonstrate that the above DNA molecules containing two copies of theCMV-luc marker gene can be encapsidated into virions, virus is harvestedfrom the remaining two cultures via three cycles of freeze-thaw crushingand is used to infect murine fibroblasts. Forty-eight hours afterinfection, the infected cells are assayed for luciferase activity. Toexclude the possibility that the luciferase activity has been induced bytransfer of free DNA, rather than via virus particles, virus stocks aretreated with DNaseI to remove DNA contaminants. Furthermore, as anadditional control, aliquots of the virus stocks are incubated for 60minutes at 56° C. The heat treatment will not affect the contaminatingDNA but will inactivate the viruses. Significant luciferase activity isonly found in the cells after infection with the virus stocks derivedfrom IG.Ad.MLPI.TK-infected cells transfected with the pICLhc and pICLIplasmids. In neither the non-infected cells nor the infected cellstransfected with the pICLhw and pICL (SEQ ID NO:21) can significantluciferase activity be demonstrated. Heat inactivation, but not DNaseItreatment, completely eliminates luciferase expression, demonstratingthat adenovirus particles, and not free (contaminating) DNA fragments,are responsible for transfer of the luciferase reporter gene.

These results demonstrate that these small viral genomes can beencapsidated into adenovirus particles and suggest that the ITR and theencapsidation signal are sufficient for encapsidation of linear DNAfragments into adenovirus particles. These adenovirus particles can beused for efficient gene transfer. When introduced into cells thatcontain and express at least part of the adenovirus genes (viz. E1, E2,E4, and L, and VA), recombinant DNA molecules that consist of at leastone ITR, at least part of the encapsidation signal, and a synthetic DNAsequence that is capable of forming a hairpin structure have theintrinsic capacity to autonomously generate recombinant genomes that canbe encapsidated into virions. Such genomes and vector system can be usedfor gene transfer.

Experiment Series 3

The following demonstrates that DNA molecules that contain nucleotides3510-35953 (viz. 9.7-100 map units) of the adenovirus type 5 genome(thus lacking the E1 protein-coding regions, the right-hand ITR, and theencapsidation sequences) and a terminal DNA sequence that iscomplementary to a portion of the same strand of the DNA molecule whenpresent in single-stranded form other than the ITR, and as a result iscapable of forming a hairpin structure, can replicate in 911 cells.

In order to develop a replicating DNA molecule that can provide theadenovirus products required to allow the above mentioned ICLhac vectorgenome and alike minimal adenovectors to be encapsidated into adenovirusparticles by helper cells, the Ad-CMV-hcTK adenoviral vector has beendeveloped. Between the CMV enhancer/promoter region and the thyrnidinekinase gene, the annealed oligonucleotide pair HP/cla1 (SEQ ID NO:19)and 2 (SEQ ID NO:20) is inserted. The vector Ad-CMV-hcTK can bepropagated and produced in 911 cell using standard procedures. Thisvector is grown and propagated exclusively as a source of DNA used fortransfection. DNA of the adenovirus Ad-CMV-hcTK is isolated from virusparticles that had been purified using CsCl density-gradientcentrifugation by standard techniques. The virus DNA has been digestedwith restriction endonuclease ClaI. The digested DNA issize-fractionated on a 0.7% agarose gel, and the large fragment isisolated and used for further experiments. Cultures of 911 cells aretransfected large ClaI-fragment of the Ad-CMV-hcTK DNA using thestandard calcium phosphate co-precipitation technique. Much like in theprevious experiments with plasmid plCLhac, the AD-CMV-hc will replicatestarting at the right-hand ITR. Once the 1-strand is displaced, ahairpin can be formed at the left-hand terminus of the fragment. Thisfacilitates the DNA polymerase to elongate the chain towards theright-hand-side. The process will proceed until the displaced strand iscompletely converted to its double-stranded form. Finally, theright-hand ITR will be recreated, and in this location the normaladenovirus replication-initiation and elongation will occur. Note thatthe polymerase will read through the hairpin, thereby duplicating themolecule. The input DNA molecule of 33250 bp, which had on one side anadenovirus ITR sequence and at the other side a DNA sequence that hadthe capacity to form a hairpin structure, has now been duplicated in away that both ends contain an ITR sequence. The resulting DNA moleculewill consist of a palindromic structure of approximately 66500 bp.

This structure can be detected in low-molecular weight DNA extractedfrom the transfected cells using Southern analysis. The palindromicnature of the DNA fragment can be demonstrated by digestion of thelow-molecular weight DNA with suitable restriction endonucleases andSouthern blotting with the HSV-TK gene as the probe. This molecule canreplicate itself in the transfected cells by virtue of the adenovirusgene products that are present in the cells. In part, the adenovirusgenes are expressed from templates that are integrated in the genome ofthe target cells (viz. the E1 gene products), the other genes reside inthe replicating DNA fragment itself. Note however, that this linear DNAfragment cannot be encapsidated into virions. Not only does it lack allthe DNA sequences required for encapsidation, but also is its size isalso much too large to be encapsidated.

Experiment Series 4

The following demonstrates that DNA molecules that contain nucleotides3503-35953 (viz. 9.7-100 map units) of the adenovirus type 5 genome(thus lacking the E1 protein-coding regions, the right-hand ITR, and theencapsidation sequences) and a terminal DNA sequence that iscomplementary to a portion the same strand of the DNA molecule otherthan the ITR, and as a result is capable of forming a hairpin structure,can replicate in 911 cells and can provide the helper functions requiredto encapsidate the pICLI- and pICLhac-derived DNA fragments.

The following series of experiments aims to demonstrate that the DNAmolecule described in Experiment Series 3 could be used to encapsidatethe minimal adenovectors described in Experiment Series 1 and 2.

In the experiments, the large fragment isolated after endonucleaseClaI-digestion of Ad-CMV-hcTK DNA is introduced into 911 cells (inconformity with the experiments described in part 1.3) together withendonuclease SalI, mungbean nuclease, endonuclease Asp718-treatedplasmid pICLhac, or, as a control, similarly treated plasmid pICLhaw.After 48 hours, virus is isolated by freeze-thaw crushing of thetransfected cell population. The virus-preparation is treated withDNaseI to remove contaminating free DNA. The virus is used subsequentlyto infect Rat2 fibroblasts. Forty-eight hours post infection, the cellsare assayed for luciferase activity. Significant luciferase activity canbe demonstrated only in the cells infected with virus isolated from thecells transfected with the pICLhac plasmid and not with the pICLhawplasmid. Heat inactivation of the virus prior to infection completelyabolishes the luciferase activity, indicating that the luciferase geneis transferred by a viral particle. Infection of 911 cell with the virusstock did not result in any cytopathological effects, demonstrating thatthe pICLhac is produced without any infectious helper virus that can bepropagated on 911 cells. These results demonstrate that the proposedmethod can be used to produce stocks of minimal adenoviral vectors thatare completely devoid of infectious helper viruses and are able toreplicate autonomously on adenovirus-transformed human cells or onnon-adenovirus transformed human cells.

Besides the system described in this application, another approach forthe generation of minimal adenovirus vectors has been disclosed inInternational Patent Publication WO 94/12649. The method described in WO94/12649 exploits the function of the protein IX for the packaging ofminimal adenovirus vectors (Pseudo Adenoviral Vectors (“PAV”) in theterminology of WO 94/12649). PAVs are produced by cloning an expressionplasmid with the gene of interest between the left-hand (including thesequences required for encapsidation) and the right-hand adenoviralITRs. The PAV is propagated in the presence of a helper virus.Encapsidation of the PAV is preferred compared with the helper virusbecause the helper virus is partially defective for packaging (either byvirtue of mutations in the packaging signal or by virtue of its size,virus genomes greater than 37.5 kb package inefficiently). In addition,the authors propose that in the absence of the protein IX gene, the PAVwill be preferentially packaged. However, neither of these mechanismsappear to be sufficiently restrictive to allow packaging of onlyPAVs/minimal vectors. The mutations proposed in the packaging signaldiminish packaging but do not provide an absolute block, as the samepackaging-activity is required to propagate the helper virus. Also,neither an increase in the size of the helper virus nor the mutation ofthe protein IX gene will ensure that PAV is packaged exclusively. Thus,the method described in WO 94/12649 is unlikely to be useful for theproduction of helper-free stocks of minimal adenovirus vectors/PAVs.

Although the application has been described with reference to certainpreferred embodiments and illustrative examples, the scope of theinvention is to be determined by reference to the appended claims.

REFERENCES

-   Berk A. J. (1986), Ann. Rev. Genet. 20, 45-79.-   Bernards R., P. I. Schrier, J. L. Bos and A. J. van der Eb (1983),    role of adenovirus types 5 and 12 early region 1b tumor antigens in    oncogenic transformation. Virology 127, 45-53.-   Bett A. J, L. Prevec and F. L. Graham (1993), Packaging Capacity and    Stability of Human Adenovirus Type-5 Vectors. J. Virol. 67,    5911-5921.-   Blaese M., T. Blankenstein, M. Brenner, O. Cohen-Hageenauer, B.    Gansbacher, S. Russell, B. Sorrentino and T. Velu (1995), vectors in    cancer therapy: how will they deliver? Cancer Gene Ther. 2, 291-297.-   Boshart M., F. Weber, G. Jahn, K. Dorsch-Häler, B. Fleckenstein    and W. Scafflier (1985), a very strong enhancer is located upstream    of an immediate early gene of human Cytomegalovirus. Cell 41,    521-530.-   Bout A., J. L. Imler, H. Schulz, M. Perricaudet, C. Zurcher, P.    Herbrink, D. Valerio and A. Pavirani (1994a), in vivo    adenovirus-mediated transfer of human CFTR cDNA to Rhesus monkey    airway epithelium: efficacy, toxicity and safety. Gene Therapy 1,    385-394.-   Bout A., M. Perricaudet, G. Baskin, J. L. Imler, B. J. Scholte, A.    Pavirani and D. Valerio (1994b), lung gene therapy: in vivo    adenovirus-mediated gene transfer to rhesus monkey airway    epithelium. Human Gene Therapy 5, 3-10.-   Brody S. L. and R. G. Crystal (1994), adenovirus-mediated in vivo    gene transfer. Ann. N.Y. Acad. Sci. 716, 90-101.-   Brough D. E., V. Cleghon and D. F. Klessig (1992), construction,    characterization, and utilization of cell lines which inducibly    express the adenovirus DNA-binding protein. Virology 190(2), 624-34.-   Brough D. E., S. A. Rice, S. Sell and D. F. Klessig (1985),    restricted changes in the adenovirus DNA-binding protein that lead    to extended host range or temperature-sensitive phenotypes. J.    Virol. 55, 206-212.-   Daniell E. (1976), genome structure of incomplete particles of    adenovirus. J. Virol. 19, 685-708.-   van der Elsen P. J., A. Houweling and A. J. van der Eb (1983),    expression of region E1B of human adenoviruses in the absence of    region E1A is not sufficient for complete transformation. Virology    128, 377-390.-   Engelhardt J. F., L. Litzky and J. M. Wilson (1994a), prolonged    transgene expression in cotton rat lung with recombinant    adenoviruses defective in E2A. Human Gene Therapy 5, 1217-1229.-   Engelhardt J. F., R. H. Simon, Y. Yang, M. Zepeda, S.    Weber-Pendleton, B. Doranz, M. Grossman and J. M. Wilson (1993),    adenovirus-mediated transfer of the CFTR gene to lung or nonhuman    primates: biological efficacy study. Human Gene Therapy 4, 759-769.-   Engelhardt J. F., X. Ye, B. Doranz and J. M. Wilson (1994b),    ablation of E2A in recombinant adenoviruses improves transgene    persistence and decreases inflammatory response in mouse liver.    Proc. Nat'l Acad. Sci. USA 91, 6196-200.-   Fang B., H. Wang, G. Gordon, D. A. Bellinger, M. S. Read, K. M.    Brinkhous, S. L. C. Woo and R. C. Eisensmith (1996), lack of    persistence of E1-recombinant adenoviral vectors containing a    temperature-sensitive E2A mutation in immunocompetent mice and    hemophilia dogs. Gene Therapy 3, 217-222.-   Fallaux F. J., O. Kranenburg, S. J. Cramer, A. Houweling, H. von    Ormondt, R. C. Hoeben and A. J. van der Eb (1996), characterization    of 911: a new helper cell line for the titration and propagation of    early region 1-deleted adenoviral vectors. Human Gene Therapy 7,    215-222.-   Gooding L. R., L. Aquino, P. J. Duerksen-Hughes, D. Day, T. M.    Horton, S. Yei and W. S. M. Wold (1991), the E1B    19,000-molecular-weight protein of group C adenoviruses prevents    tumor necrosis factor cytolysis of human cells but not of mouse    cells. J. Virol. 65, 3083-3094.-   Gräble M. and P. Hearing (1990), adenovirus type 5 packaging domain    is composed of a repeated element that is functionally redundant. J.    Virol. 64, 2047-2056.-   Gräble M. and P. Hearing (1992), cis and trans Requirements for the    Selective Packaging of Adenovirus Type-5 DNA. J. Virol. 66, 723-731.-   Graham F. L. and A. J. van der Eb (1973), a new technique for the    assay of infectivity of human adenovirus 5 DNA. Virology 52,    456-467.-   Graham F. L., J. Smiley, W. C. Russell and R. Naira (1977),    characteristics of a human cell line transformed by DNA from    adenovirus type 5. J. Gen. Virol. 36, 59-72.-   Haddada H., T. Ragot, L. Cordier, M. T. Duffour and M. Perricaudet    (1993), adenoviral interleukin-2 gene transfer into P815 tumor cells    abrogates tumorigenicity and induces antitumoral immunity in mice.    Human Gene Therapy 4, 703-11.-   Hay R. T., N. D. Stow and I. M. McDougall (1984), replication of    adenovirus minichromosomes. J. Mol. Biol. 174, 493-510.-   Hearing P., R. J. Samulski, W. L. Wishart and T. Shenk (1987),    identification of a repeated sequence element required for efficient    encapsidation of the adenovirus type 5 chromosome. J. Virol. 61,    2555-2558.-   Horwitz M. S. (1990), Adenoviridae and Their Replication, pp.    1679-1740, in B. N. Fields and D. M. Knipe (Eds), Virology, Raven    Press, Ltd, New York.-   Hu C. H., F. Y. Xu, K. Wang, A. N. Pearson and G. D. Pearson (1992),    Symmetrical Adenovirus Minichromosomes Have Hairpin Replication    Intermediates. Gene 110, 145-150.-   Imler J. L., C. Chartier, D. Dreyer, A. Dieterle, M.    Sainte-Marie, T. Faure, A. Pavirani and M. Mehtali (1996), novel    complementation cell lines derived from human lung carcinoma A549    cells support the growth of E1-deleted adenovirus vectors. Gene    Ther. 3, 75-84.-   Jochemsen A. G., L. T. C. Peltenburg, M. F. W. T. Pas, C. M. de    Wit, J. L. Bos and A. J. van der Eb (1987). EMBO J. 6,3399-3405.-   Klessig D. F. and T. Grodzicker (1979), mutations that allow human    Ad2 and Ad5 to express late genes in monkey cells maps in the viral    gene encoding the 72K DNA-binding protein. Cell 17, 957-566.-   Klessig D. F., T. Grodzicker and V. Cleghon (1984), construction of    human cell lines which contain and express the adenovirus DNA    binding protein gene by co-transformation with the HSV-1 tk gene.    Virus Res. 1, 169-188.-   Kruijer W., J. C. Nicolas, F. M. von Schaik and J. S. Sussenbach    (1983), structure and function of DNA binding proteins from    revertants of adenovirus type 5 mutants with a temperature-sensitive    DNA replication. Virology 124, 425-433.-   Lechner R. L. and T. J. Kelly Jr. (1977), the structure of    replicating adenovirus 2 DNA molecules. J. Mol. Biol. 174, 493-510.-   de Leij L., P. E. Postmus, C. H. C. M. Buys, J. D. Elema, F.    Ramaekers, S. Poppema, M. Brouwer, A. Y. van der Veen, G. Mesander    and T. H. The (1985), characterization of three new variant type    cell lines derived from small cell carcinoma of the lung. Cancer    Res. 45, 6024-6033.-   Levrero M., V. Barban, S. Manteca, A. Ballay, C. Balsamo, M. L.    Avantaggiati, G. Natoli, H. Skellekens, P. Tiollais and M.    Perricaudet (1991), defective and nondefective adenovirus vectors    for expressing foreign genes in vitro and in vivo. Gene 101,    195-202.-   Lochmüller H., A. Jani, J. Huard, S. Prescott, M. Simoneau, B.    Massie, G. Karpati and G. Acasdi (1994), emergence of early region    1-containing replication-competent adenovirus in stocks of    replication-defective adenovirus recombinants (ΔE1-ΔE3) during    multiple passages in 293 cells. Hum. Gene Ther. 5, 1485-1492.-   Matsui T., M. Murayama and T. Mita (1986), adenovirus 2 peptide IX    is expressed only on replicated DNA molecules. Mol. Cell Biol. 6,    4149-4154.-   Michelson A. M., A. F. Markham and S. H. Orkin (1983), isolation and    DNA sequence of a full-length cDNA clone for human    X-chromosome-encoded phosphoglycerate kinase. Proc. Nat'l. Acad.    Sci. USA 80, 472-476.-   Morin J. E., M. D. Lubeck, J. E. Barton, A. J. Conley, A. R. Davis    and P. P. Hung (1987), recombinant adenovirus induces antibody    response to hepatitis B virus surface antigens. Proc. Nat'l. Acad.    Sci. USA, 84, 4626-4630.-   Nicolas J. C., F. Suarez, A. J. Levine and M. Girard (1981),    temperature-independent revertants of adenovirus H5ts125 and H5ts107    mutants in the DNA binding protein: isolation of a new class of host    range temperature conditional revertants. Virology 108, 521-524.-   Ostrove J. M. (1994), safety testing programs for gene therapy viral    vectors. Cancer Gene Therapy 1, 125-131.-   Pacini D. L., E. J. Dubovi and W. A. Clyde (1984), J. Infect. Dis.    150, 92-97.-   Postmus P. E., L. de Ley, A. Y. van der Veen, G.    Mesander, C. H. C. M. Buys and J. D. Elema, (1988), two small cell    lung cancer cell lines established from rigid bronchoscope biopsies.    Eur. J Clin. Oncol. 24, 753-763.-   Rice S. A. and D. F. Klessig (1985), isolation and analysis of    adenovirus type 5 mutants containing deletions in the gene encoding    the DNA-binding protein. J. Virol. 56, 767-778.-   Roberts B. E., J. S. Miller, D. Kimelman, C. L. Cepko, I. R.    Lemischka and R. C. Mulligan (1985), J. Virol. 56, 404-413.-   Shapiro D. L., L. L. Nardone, S. A. Rooney, E. K. Motoyama and J. L.    Munoz (1978), phospholipid biosynthesis and secretion by a cell line    (A549) which resembles type II alveolar epithelial cells. Biochim.    Biophys. Acta. 530, 197-207.-   Simon R. H., J. F. Engelhardt, Y. Yang, M. Zepeda, S.    Weber-Pendleton, M. Grossman and J. M. Wilson (1993),    adenovirus-mediated transfer of the CFTR gene to lung of nonhuman    primates: toxicity study. Human Gene Therapy 4, 771-780.-   Singer-Sam J., D. H. Keith, K. Tani, R. L. Simmer, L. Shively, S.    Lindsay, A. Yoshida and A. D. Riggs (1984), sequence of the promoter    region of the gene for X-linked 3-phosphoglycerate kinase. Gene 32,    409-417.-   Stein R. W. and J. Whelan (1989), insulin gene enhancer activity is    inhibited by adenovirus 5 E1A gene products. Mol. Cell Biol. 9,    4531-4.-   Stratford-Perricaudet L. D. and M. Perricaudet (1991), Gene Transfer    into Animals: the Promise of Adenovirus, pp. 51-61, in O.    Cohen-Adenauer and M. Boiron (Eds). Human Gene Transfer, John Libbey    Eurotext.-   Telling G. C., S. Perera, O. M. Szatkowski and J. Williams (1994),    absence of an essential regulatory influence of the adenovirus E1B    19-kilodalton protein on viral growth and early gene expression in    human diploid W138, HeLa, and A549 cells. J. Virol. 68, 541-7.-   Tooze J. (1981), DNA Tumor Viruses (revised), Cold Spring Harbor    Laboratory, Cold Spring Harbor, N.Y.-   Vieira J. and J. Messing (1987), Production of single-stranded    plasmid DNA, pp. 3-11, Methods in Enzymology, Acad. Press Inc.-   Vincent A. J. P. E., M.d.C. Esandi, G. D. von Someren, J. L.    Noteboom, C. J. J Avezaat, C. Vecht, P. A. E. S. Smitt, D. W. von    Bekkum, D. Valerio, P. M. Hoogerbrugge and A. Bout (1996a),    treatment of Lepto-meningeal metastasis in a rat model using a    recombinant adenovirus containing the HSV-tk gene. J. Neurosurg. in    press.-   Vincent A. J. P. E., R. Vogels, G. von Someren, M.d.C. Esandi, J. L.    Noteboom, C. J. J. Avezaat, C. Vecht, D. W. von Bekkum, D.    Valerio, A. Bout and P. M. Hoogerbrugge (1996b), Herpes Simplex    Virus Thymidine Kinase gene therapy for rat malignant brain tumors.    Hum. Gene Ther. 7, 197-205.-   Wang K. and G. D. Pearson (1985), adenovirus sequences required for    replication in vivo. Nucl.

Acids Res. 13,5173-5187.

-   White E., A. Denton and B. Stillman (1988), J. Virol. 62, 3445-3454.-   Yang Y., Q. Li, H. C. J. Ertl and J. M. Wilson (1995), cellular and    humoral immune responses viral antigens create barriers to    lung-directed gene therapy with recombinant adenoviruses. J. Virol.    69, 2004-2015.-   Yang Y., F. A. Nunes, K. Berencsi, E. E. Furth, E. Gonczol and J. M.    Wilson (1994a), cellular immunity to viral antigens limits    E1-deleted adenoviruses for gene therapy. Proc. Nat'l. Acad. Sci.    USA 91, 4407-11.-   Yang Y., F. A. Nunes, K. Berencsi, E. Gonczol, J. F. Engelhardt    and J. M. Wilson (1994b), inactivation of E2A in recombinant    adenoviruses improves the prospect for gene therapy in cystic    fibrosis. Nature Genetics 7, 362-9.-   Zantema A., J. A. M. Fransen, A. Davis-Olivier, F. C. S.    Ramaekers, G. P. Vooijs, B. Deleys and A. J. van der Eb (1985),    localization of the E1B proteins of adenovirus 5 in transformed    cells, as revealed by interaction with monoclonal antibodies.    Virology 142, 44-58.

1. An isolated first nucleic acid molecule comprising geneticinformation encoding functional adenovirus E1A, E1B 21 kDa and E1B 55kDa gene products, wherein said first nucleic acid molecule lacksgenetic information from an adenovirus pIX gene that can mediatehomologous recombination with a second nucleic acid molecule havinggenetic information encoding a functional or active adenovirus pIX geneproduct, and further wherein said first nucleic acid molecule lacksgenetic information encoding adenovirus proteins other than proteinsencoded by adenovirus early region
 1. 2. The isolated first nucleic acidmolecule of claim 1, wherein the isolated nucleic acid moleculecomprises nucleotides 459-3510 of Ad5.
 3. The isolated first nucleicacid molecule of claim 2, wherein the isolated nucleic acid moleculelacks nucleotides from the E1 region of Ad5 downstream of nucleotide3510.
 4. The isolated first nucleic acid molecule of claim 1, furthercomprising a promoter operatively linked to at least the geneticinformation encoding the functional E1A gene product.
 5. The isolatedfirst nucleic acid molecule of claim 4, wherein the promoter comprises aconstitutive promoter.
 6. The isolated first nucleic acid molecule ofclaim 5, wherein the constitutive promoter comprises a PGK promoter. 7.The isolated first nucleic acid molecule of claim 4, wherein thepromoter comprises a human PGK promoter.
 8. The isolated first nucleicacid molecule of claim 1, further comprising the Hepatitis B Viruspolyadenylation signal located downstream of an E1B stop codon.
 9. Theisolated first nucleic acid molecule of claim 4, further comprising theHepatitis B Virus polyadenylation signal located downstream of an E1Bstop codon. 10.-18. (canceled)
 19. An isolated adenovirus packaging cellcomprising the isolated first nucleic acid molecule of claim
 1. 20. Theisolated adenovirus packaging cell of claim 19, wherein the cell is amammalian cell.
 21. The isolated adenovirus packaging cell of claim 19,wherein the cell is a human cell.
 22. The isolated adenovirus packagingcell of claim 19, wherein the cell is a diploid cell.
 23. The isolatedadenovirus packaging cell of claim 19, wherein the cell is derived froman embryonic cell.
 24. The isolated adenovirus packaging cell of claim23, wherein the embryonic cell is selected from the group consisting ofkidney cells, lung cells, and retinoblast cells.
 25. The isolatedadenovirus packaging cell of claim 24, wherein the embryonic cell is aretinoblast cell.
 26. The isolated adenovirus packaging cell of claim19, wherein the cell is immortalized.
 27. A method for making anisolated adenovirus packaging cell comprising introducing into the cellthe isolated first nucleic acid molecule of claim
 1. 28. The methodaccording to claim 27, further comprising integrating the isolated firstnucleic acid molecule into the precursor cell's genome. 29.-37.(canceled)